What is Fucci?

Fluorescent ubiquitination-based cell cycle indicator (Fucci) is a sophisticated technology which can easily determine G1 and/or S/G2/M phases of the cell cycle. The technology analyzes living cells in a spatio-temporal manner using a dual color scheme of orange and green.
Fucci was successfully established by intelligently utilizing ubiquitin-proteasome protein degradation system (awarded the honor of "Top Innovations of 2008”).

Properties

Schematic representation of Fucci constructs

Fucci is a set of fluorescent probes: Fucci-G1 Orange and Fucci-S/G2/M Green.
Fucci-G1 Orange is a fusion protein of a fragment of human Cdt1 (amino acids 30-120) with the orange fluorescent mKO2 (monomeric Kusabira-Orange2) that indicates the G1 phase. Fucci-S/G2/M Green is a fusion protein of a fragment of human Geminin (amino acids 1-110) with the green fluorescent protein mAG1 (monomeric Azami-Green1) that visualizes S, G2 and M phases. Fucci-S/G2/M Green (N+C) encodes a fusion protein of a part of human Geminin (1-60) with mAG1.

Terms

Cdt1 :Cdc10 dependent transcript 1 is a conserved replication factor required for licensing the chromosome for a single course of DNA synthesis. Abundantly expressed throughout the cell cycle, Cdt1 is ubiquitinated by the ubiqutin ligase complex SCF skp2 during S and G2 phases and degraded by proteasome.

Geminin: Geminin inhibits the licensing activity of Cdt1. Geminin interferes with the binding of licensing factors to the replication origin once a chromosome has started to replicate during the S phase. During M and G1 phases, Geminin is ubiquitinated by the E3 ligase complex APCcdh1 and degraded by the proteasome.

Fluorescence characteristics

The technology was established by using Amalgaam’s proprietary fluorescent proteins: mAG1 (monomeric Azami-Green1) and mKO2(monomeric Kusabira-Orange2). Both are bright fluorescent proteins and mature rapidly. The graph above shows excitation (dotted line) and emission (solid line) spectra of these proteins.

Provided by Dr. Sakaue-Sawano at RIKEN.
Cell. (2008) 132:487-98.

Excit./Emiss.Maxima (nm)

Extinction coefficient (M-1cm-1)

Fluorescence quantum yield

pH sensitivity

mAG1

492/505

55,500 (492 nm)

0.74

pKa=5.8

mKO2

551/565

63,800 (551 nm)

0.62

pKa=5.5

Cell cycle regulation by E3 ligase

The cell cycle is controlled by ubiqutin-mediated proteolysis. The APCCdh1 (anaphase promoting complex) and SCFSkp2 (SKP-Cullin-F-box) complexes are E3 ligase activities that modify the corresponding target proteins with ubiquitin in a cell cycle-dependent manner. The APCCdh1 complex is active in the late M and G1 phases, while the SCFSkp2 complex is active in the S and G2 phases. The substrates of the APCCdh1 and SCFSkp2 complexes are Geminin and Cdt1 respectively.
In the Fucci system, the phases in which Geminin or Cdt1 are not degraded by the proteasome system can be visualized in living cells.

Analysis

Fluorescent images of Fucci cells

Each cell cycle of the G1, G1/S , S, G2, and M phases can be determined by the combination of Fucci-G1 Orange, Fucci-S/G2/M Green, and an antibody against PCNA.
G1 phase is indecated by orange. Both orange and green were observed in the G1/S phase. Additional immunostaining color by PCNA was observed at the initiation of the S phase. Cells with pure green fluorescence were either in the S or G2 phase and were distinguished by immunostaining of the S phase. The rest of the cells were classified into the M phase.
These results are consistent with the fact that Cdt1 accumulates in G1, while Geminin accumulates in S/G2/M. PCNA: Proliferation Cell Nuclear Antigen.

Provided by Dr. Sakaue-Sawano at RIKEN.
Cell. (2008) 132:487-98.

Analysis by flow cytometry

Fucci-expressing HeLa cells were analyzed using BD LSR (Becton Dickinson).
Both mKO2 and mAG were excited by a single 488 nm laser line (Ar). Fluorescence signals were collected at 530 nm (530/28 BP)(FL1) for mAG and at 575 nm (575/26 BP)(FL2) for mKO2. The data were analyzed using FlowJo software (Tree Star).

Provided by Dr. Sakaue-Sawano at RIKEN.
Cell. (2008) 132:487-98.

Cell cycle dependent changes in fluorescence

1/110 and 1/60 Fucci Green

The difference between these two green probes is the length of the domain.
Fucci-S/G2/M Green consists of the amino acids from 1 to 110, while (N+C) is from 1 to 60. (N+C) can visualize not only the cell nucleus but also cytoplasm.
We believe that the greatest advantage of the (N+C) probe is the ability to obtain not only the information of the cell cycle but also of the cell morphology.

■To establish stable cell lines expressing Fucci G1 Orange or Fucci S/G2/M Green, the efficiency of establishment increases by transfecting each genes by expressing vectors which have different drug resistance genes from each other.
Check the PDF for the detailed protocol.PDF: 96KB

※CoralHue® fluorescent proteins were co-developed with the Laboratory
for Cell Function and Dynamics, the Advanced Technology Development
Center, the Brain Science Institute, RIKEN. MBL possesses the license
and deals in the products.

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